Toxoplasma gondii is an extremely successful intracellular pathogen that causes lifelong chronic infections in almost all warm-blooded animals and severe disease in fetuses and immunocompromised individuals. The Lewis rat is the only known animal that has sterilizing immunity against the parasite and is therefore a unique animal model that can help us understand how the immune system can defeat T.gondii. NLRP1 inflammasome activation in Lewis rat macrophages upon T.gondii infection induces pyroptosis resulting in loss of the parasite?s replication niche and clearance of infection. The mechanism by which NLRP1 inflammasomes detect T.gondii infection and become activated remains poorly understood. The murine NLRP1 inflammasome is activated by ubiquitination and proteasomal degradation of its auto-inhibitory N-terminal polypeptide, which liberates the C-terminal polypeptide for Caspase-1 recruitment and inflammasome assembly. Therefore, E3 ubiquitin ligases from pathogens and/or host play a critical role in ubiquitination of NLRP1 and inflammasome activation. We previously identified three T.gondii secreted effector proteins that are required for NLRP1 inflammasome activation in Lewis rat macrophages, however, none of these effectors possess predicted ubiquitin ligase function. Immunoprecipitation identified several host E3 ubiquitin ligases that interact with these T.gondii effectors. In addition, we have found that the murine NLRP1 inflammasome activator, Val-boroPro, an inhibitor of the cytosolic serine dipeptidase DPP8/9, induces pyroptosis in Lewis rat macrophages but not in macrophages from rat strains resistant to Toxoplasma-induced pyroptosis. Thus, Val-boroPro phenocopies T.gondii infection in Lewis macrophages. Murine NLRP1 inflammasome activation triggered by inhibition of DPP8/9 requires ubiquitination of NLRP1 via unknown E3 ubiquitin ligases. Thus, we hypothesize that the Lewis E3 ubiquitin ligases interacting with our T.gondii effectors, or other host E3 ubiquitin ligases, serve as checkpoints for NLRP1 inflammasome activation in Lewis rat macrophages in response to T.gondii infection and DPP8/9 inhibition. This hypothesis will be tested by pursuing two specific aims. In our first aim we will evaluate the role of the Lewis rat E3 ubiquitin ligases interacting with our T.gondii effectors in T.gondii- and Val-boroPro-induced pyroptosis of Lewis rat macrophages, and in binding to and ubiquitination of Lewis rat NLRP1. In our second aim we will use an unbiased gain-of-function CRISPR/Cas9 screen to identify the host E3 ubiquitin ligases involved in Lewis rat NLRP1 inflammasome activation triggered by T.gondii infection and DPP8/9 inhibition. This proposal will advance the field by: 1) identifying the mechanism by which T.gondii activates the Lewis rat NLRP1 inflammasome, which could lead to novel insights into controlling toxoplasmosis in humans as human NLRP1 polymorphisms are associated with severity of congenital toxoplasmosis; 2) uncovering new principles and mechanisms regulating the innate immune system including pathogen recognition and inflammasome activation.